This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mentor: Dr. Jeffrey Cirillo (Texas A&M) Specific Aims: Quorum sensing (QS) is a bacterial communication system that utilizes secreted chemical molecules in a density dependent manner. This type of communication system regulates a variety of cellular functions and has been shown to play a role in bacterial pathogenesis (Xavier and Bassler, 2005;Zhang and Dong, 2004). The specific aim in this study is to determine if QS occurs in mycobacteria and the possible role QS plays in mycobacterial pathogenesis. The bacterial species Streptomyces has a well-defined QS system and its QS molecule has been identified. Both Streptomyces and Mycobacterium are gram-positive Actinomycetales, have a G-C rich genome and reside in the soil, therefore, it is likely that similar QS systems exists in Mycobacterium. The hypothesis for this study is that a QS system exists in Mycobacterium. Streptomyces will be used as a model organism to study QS in Mycobacterium. This research design is aimed at isolating a biologically active QS molecule, determining the structure of the isolated QS molecule and identifying gene(s) involved in QS in Mycobacterium. Background and Significance: Quorum sensing (QS) is a type of communication between cells in a density-dependent manner. QS plays a role in a variety of cellular functions such as gene expression, gene transfer, sporulation, virulence, and antibiotic production. Many quorum sensing molecules have been found in different types of bacteria. The chemicals involved in quorum sensing are also referred to as autoinducers and allow for intraspecies communication (Xavier and Bassler, 2005). The process of QS allows bacteria to determine the numbers of identical bacteria in a population and thereby alter gene expression synchronously in the population. It has been found that QS-controlled processes are important for successful bacterial-host relationships, including symbiotic and pathogenic relationships (Xavier and Bassler, 2005). The nature and function of these QS molecules are diverse. Gram-negative bacteria such as Pseudomonas aeruginosa and Vibrio fisheri use low molecular weight substances such as N-acyl homoserine lactones (AHLs) as pheromones. Some of the functions of AHLs include conjugation, virulence enzyme production, biofilm production, bioluminescence, and motility. Gram-positive bacteria such as Bacillus subtilis, Myxococcus sp., and Streptomyces sp. use a variety of different signaling molecules that include oligopeptides, fatty acids, and butyrolactones, respectively. Processes that are regulated by QS molecules in gram-positive bacteria include sporulation, fruiting body formation, virulence, and antibiotic production (Volosin and Kaprelyants, 2004). QS has been found to play a significant role in pathogenesis in a variety of different bacterial species. Candida albicans and Pseudomonas aeruginosa are two examples. In C. albicans, the QS molecule called farnesol has been shown to suppress mycelium formation. The transition from the yeast form to the mycelium form of C. albicans is essential for its pathogenicity and if this transition can be prevented then this human pathogen could be better controlled. Development of farnesol analogs could prove to have clinical significance and could hold great potential for sufferers of Candidiasis, which occurs when C. albicans is able to adhere, colonize and invade epithelial tissues (Shchepin, Hornby, Bruger, Niessen, Dussault, and Nickerson, 2003). Pseudomonas aeruginosa, a gram-negative opportunistic human pathogen, also utilizes QS for pathogenesis and as stated earlier utilizes N-acyl homoserine lactone (AHL) as the signaling molecule. This pathogen is a common cause of nosocomial infections and a major cause of lung infections in cystic fibrosis patients. Key to the expression of many of the virulence factors found in Pseudomonas is density-dependent gene regulation. Some examples of genes expressed due to QS in Psuedomonas exoenzymes, toxins and genes needed for the development of biofilms. To emphasize the density-dependent method used by Psuedomonas it has been shown that quorum sensing molecules are produced in higher quantities late in the exponential growth phase and that once the population of bacteria reach a critical density, the bacteria are capable of overwhelming the host and sabotaging host defenses (Arevalo-Ferro, Hentzer, Reil, Gorg, Kjelleberg, Givskov, Riedel and Eberl, 2003). Due to the role of QS in pathogenesis, it is hypothesized that Mycobacterium also possesses a QS system. The specific aim in this study is to determine if QS occurs in Mycobacterium and the possible role QS plays in mycobacterial pathogenesis. In this study the non-pathogenic mycobacterial species, Mycobacterium smegmatis, will be used to study this phenomenon. The information obtained from M. smegmatis may be applicable to Mycobacterium tuberculosis and help to identify if QS plays a role in pathogenesis. The information obtained may be useful in determining how M. tuberculosis is able to overcome host defenses and help to identify a means to treat and/or prevent tuberculosis. Mycobacterium smegmatis, was chosen in this experiment for specific reasons. First, unlike many other species of bacteria from this genus, M. smegmatis is non-pathogenic. This makes it suitable and safe for human handling, essential for laboratory work at an undergraduate institution such as Nebraska Wesleyan University. Second, because it is in the same genus as pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae, its quorum-sensing mechanism is most likely very similar to that of these virulent strains (El-Etr, Subbian, S. Cirillo, and J. Cirillo, 2004). If a deeper understanding of M. smegmatis can be developed, it can serve as a model for M. tuberculosis and M. leprae. In turn, providing insight into the disease-causing mechanism of these pathogenic strains, thereby holding value for humankind. One of the most thoroughly studied quorum sensing systems is that of Streptomyces. Streptomyces is known to produce many secondary metabolites, many involved in morphological differentiation such as development of aerial mycelium and sporulation (Choi, Lee, Hwang, Kinosita, and Hihira, 2003). In the early 90's A-factor (2S-isocapryloyl-3S-hydroxymethyl-g-butyrolactone) was discovered as an autoregulator in Streptomyces griseus, and its release caused the production of streptomycin, induction of aerial hyphae and pigmentation (Horinouchi and Beppu, 1992;Takano, Nihira, Hara, Jones, Gershater, Yamada, and Bibb, 2000). Mutants of Streptomyces unable to produce A-factor also lack the ability to undergo sporulation, make pigments, or antibiotics. Both Streptomyces and Mycobacterium are gram-positive bacteria with a high G-C content in their genome. They both belong to the phylum, Actinomycetales, and both live in the soil, suggesting that they come in contact with one another and have the ability to respond to one another. Because quorum sensing assays are well established for Streptomyces and the signaling molecules identified, Streptomyces will serve as a useful tool in the study of QS in Mycobacterium and be utilized to identify QS molecules in Mycobacterium. It is hypothesized that Mycobacterium smegmatis does possess a QS system and preliminary results in my laboratory indicate that a biologically active molecule isolated from Mycobacterium smegmatis does elicit a response (aerial mycelia formation and pigmentation) in Streptomyces. Research Design and Methods: The proposed project has two main objectives. The first objective is to isolate a biologically active QS molecule from Mycobacterium smegmatis using ethyl acetate extractions. The extracted molecule will be tested on lawns of Streptomyces and a biologically active extract will show increased pigmentation and/or aerial hyphae on lawns of Streptomyces. Once a biologically active molecule has been obtained, the molecule will be purified using HPLC, and the structure of the QS molecule determined using Mass Spectrometry. The second objective is to identify gene(s) involved in QS in Mycobacterium smegmatis. To accomplish this, two Mycobacterium smegmatis libraries will be used. The first is an overexpression library containing Mycobacterium marinum genes inserted into Mycobacterium smegmatis and the second library will be a library of Mycobacterium smegmatis mutants obtained by transposon mutagenesis. Clones from both libraries will be screened for increased or diminished arial hyphae and/or pigmentation when tested against lawns of Streptomyces. Positive clones will be analyzed further and DNA from these clones will be isolated and sequenced to determine which gene has been disrupted (transposon mutagenesis library) or is present in duplicate (overexpression library). Preliminary Work Performed and Results: 1. Development of QS Assay to test if Mycobacterium smegmatis secretes a biogically active QS molecule that Streptomyces can respond to. a. To test if Mycobacterium smegmatis secretes a molecule that Streptomyces coelicolor can respond to, the indicator strain (Streptomyces coelicolor M145) was streaked on one half of a Petri plate and the test strain (Mycobacterium smegmatis) was streaked on the other half of the plate. If Mycobacterium smegmatis secretes a molecule that Streptomyces coelicolor responds to, Streptomyces coelicolor will exhibit accelerated pigmentation and/or aerial hyphae formation. b. Results obtained [unreadable]Streptomyces coelicolor responds to a molecule secreted by Mycobacterium smegmatis as seen in Figure 1. Aerial hyphae production and pigmentation of Streptomyces coelicolor in response to Mycobacterium smegmatis was observed.